Acetylation of p53 Protein Is Crucial for Its Function In Myeloid Cells.

Abstract 1480

Severe congenital neutropenia (CN) is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 10⁹/L. G-CSF treatment increases blood neutrophil counts in more than 90% of individuals with CN. CN is also considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop Acute Myelocytic Leukemia (AML) or Myelodysplastic Syndrome (MDS). Surprisingly no mutations in genes, which typically occur in primary AML/MDS patients, were detected in CN patients who developed leukemia. But studies in CN patients reveal a high association of G-CSF receptor mutation and the incidence of leukemia, indicating the dysregulation of certain factors downstream of G-CSF receptor signalling. Recently, we reported that Nicotinamide Phosphoribosyltransferase (NAMPT), a protein involved in biosynthesis of NAD⁺, was significantly increased in CN patients treated with G-CSF as compared to healthy individuals. Elevated NAMPT/NAD⁺ levels correlated with increased levels of SIRT1, a NAD⁺-dependent deacetylase which is involved in the deacetylation of histone and non-histone proteins e.g. p53. The acetylation of tumor-suppressor p53 is considered necessary for its transcriptional activation, while SIRT1-mediated deacetylation of p53 has been shown to attenuate the transcriptional activity of p53. Therefore, we asked if deacetylation-dependent inactivation of p53 might play a role in leukemic transformation in CN patients. In this study we demonstrate that the presence of NAMPT or NAD⁺ enhances the SIRT1-mediated deacetylation of p53 in both the 293T cell line and the promyelocytic leukemia NB4 cell line. Treatment with exogenous recombinant NAMPT also leads to a decrease in the acetylation levels of endogenous p53 in CD34⁺ cells. The cyclin-dependent kinase inhibitor 1A (p21, Cip1) protein is a well-known target of p53 and is involved in cell cycle arrest. We have shown that over-expression of NAMPT leads to down-regulation of p21 mRNA, and specific knockdown of SIRT1 leads to up-regulation of p21 mRNA. The presence of NAMPT also decreases the mRNA levels of p21 in both NB4 and CD34⁺ cells. The compound FK866 specifically inhibits NAMPT and has recently entered clinical trials as a potential chemotherapeutic agent. In a recent preclinical in vitro study FK866 has been shown to elicit massive cell death in numerous leukemia/lymphoma cell lines, but the underlying molecular mechanism remains unknown. We tested if inhibition of NAMPT using FK866 enhances the tumor-supressing role of p53 by increasing its acetylation levels. We have demonstrated that the treatment of NB4 cells with FK866 increases the acetylation of endogenous p53, and this increased acetylation is in part due to decreased interaction of p53 with SIRT1. In addition, the mRNA levels of p21 down-regulated in CD34⁺ and NB4 cells on treatment with NAMPT were up-regulated on use of FK866. Knockdown of p53 using specific shRNA against p53 inhibits the expression of p21 and treatment with FK866 under p53 knockdown does not induce the expression of p21 when compared to control cells. In functional studies we show that over-expression of NAMPT leads to increased proliferation of both 293T and NB4 cells. Treatment with FK866 leads to increased death of NB4 cells compared to the cells in which p53 has been silenced, due to the lack of p53 available to be acetylated. Taken together, our conclusion is that NAMPT/NAD⁺ activated SIRT1 mediates deacetylation of p53 leading to down-regulation of the downstream target gene p21. This inhibition of the tumor-suppressor functions of p53 might be involved in the leukemic transformation seen in CN.